KR102255902B1 - A method for regulating galactosylation of recombinant proteins by optimizing culture media - Google Patents

Abstract

The present invention comprises the step of raising the osmotic pressure of the animal cell culture medium during the culturing process of the animal cell expressing the desired recombinant protein, galactosylation (galactosylation) is regulated, the production method of the desired recombinant protein or the desired recombinant A method for controlling the galactoseization of a protein, comprising the step of supplementing asparagine to the animal cell culture medium during the cultivation of an animal cell expressing the desired recombinant protein, a method for producing a recombinant protein of interest, in which galactosization is regulated, or A method of regulating galactoseization of a recombinant protein of interest and raising the osmotic pressure of the animal cell culture medium during culturing of an animal cell expressing the desired recombinant protein, and supplementing asparagine, wherein galactoseization is regulated, It relates to a method for preparing a recombinant protein of interest or a method for controlling galactoseization of a recombinant protein of interest, and a recombinant protein of interest in which galactosization prepared by the method is regulated.

Description

{A method for regulating galactosylation of recombinant proteins by optimizing culture media}

The present invention comprises the step of raising the osmotic pressure of the animal cell culture medium during the culturing process of the animal cell expressing the desired recombinant protein, galactosylation (galactosylation) is regulated, the production method of the desired recombinant protein or the desired recombinant A method of regulating the galactoseization of proteins; A method for producing a recombinant protein of interest, comprising the step of supplementing asparagine to the animal cell culture medium during the culturing process of an animal cell expressing the desired recombinant protein, or regulating the galactoseization of the desired recombinant protein How to; In the process of culturing animal cells expressing the desired recombinant protein, a method for producing a recombinant protein of interest or a method for producing a recombinant protein of interest, comprising the step of increasing the osmotic pressure of the animal cell culture medium and supplementing asparagine A method of regulating the galactosization of; And it relates to a recombinant protein of interest prepared by the above method, the galactose is regulated.

Antibodies are proteins that can bind antigens to interfere with the function of antigens or remove antigens.As the therapeutic antibody market has been found to have considerable potential, studies for efficient expression and mass production of antibodies are actively progressing. have. One of the important points in the production of such antibodies is the homogeneity of the produced antibody population. In particular, in the production of monoclonal antibodies, the glycoform profile of a constant and reproducible monoclonal antibody is important. However, various variables during the cell culture process can affect the glycosylation profile of the monoclonal antibody produced. Accordingly, there has been a demand for the development of a method capable of uniformly controlling the glycosylation of the produced antibody population and the content of isomeric antibodies.

One of the important challenges in the commercial production process of monoclonal antibodies is to consistently maintain the quality of monoclonal antibodies produced from batch to batch. The qualities to be checked during the production process of monoclonal antibodies are numerous, but galactoseization of antibodies can be seen as one of the most important among them. This is because galactose is known to affect the CDC (complement dependent cytotoxicity) process, one of the largest mechanisms of action (MOA) of monoclonal antibodies. The galactosylation is achieved by using galactose as a constituent unit of a glycosylation chain reaction, and attaching it to the N-acetylglucosamine sugar through a galactosyltransferase. As a method for controlling galactosylation, a method of adding manganese or galactose to the culture medium has been proposed (US Patent Publication No. 2012-0276631), but development of a method for controlling galactosylation of antibodies is still required. do.

Under this background, the present inventors made diligent efforts to develop a method that can effectively control the galactosylation of the recombinant protein produced in the cultivation process of the animal cell producing the recombinant protein. As a result, during the cultivation process of the animal cell expressing the recombinant protein. It was confirmed that when the osmotic pressure of the animal cell culture solution was increased or asparagine was supplemented at a specific culture point, galactosylation of the produced recombinant protein could be effectively prepared. In addition, it was confirmed that when asparagine was supplemented with an increase in the osmotic pressure of the animal cell culture solution, galactosylation could be controlled without a decrease in the content of the acidic isomer antibody due to the increase in the osmotic pressure, thereby completing the present invention.

One object of the present invention is a method for producing a recombinant protein of interest, in which galactosylation is regulated, comprising raising the osmotic pressure of the animal cell culture medium during the culturing process of an animal cell expressing the desired recombinant protein. Is to provide.

Another object of the present invention is to provide a method for regulating galactoseization of a recombinant protein of interest, comprising raising the osmotic pressure of the animal cell culture medium during the cultivation of animal cells expressing the desired recombinant protein.

Another object of the present invention is to provide a method for producing a recombinant protein of interest, in which galactose is regulated, comprising the step of supplementing asparagine to the animal cell culture medium during the culturing process of an animal cell expressing the desired recombinant protein. will be.

Another object of the present invention is to provide a method for regulating galactoseization of a recombinant protein of interest, comprising the step of supplementing asparagine to the animal cell culture medium during the cultivation of animal cells expressing the desired recombinant protein. .

Another object of the present invention is to increase the osmotic pressure of the animal cell culture medium during the cultivation of animal cells expressing the desired recombinant protein, and supplementing asparagine. It is to provide a manufacturing method.

Another object of the present invention is to increase the osmotic pressure of the animal cell culture medium during the culturing process of the animal cell expressing the desired recombinant protein, and to control the galactoseization of the desired recombinant protein, comprising the step of supplementing asparagine. Is to provide a way.

Another object of the present invention is to provide a recombinant protein of interest prepared by the above method.

One aspect of the present invention for achieving the above object is to increase the osmotic pressure of the animal cell culture medium during the culturing process of the animal cell expressing the desired recombinant protein. It is a manufacturing method of.

Specifically, the method is a method for producing a recombinant protein of interest, in which galactose is regulated, comprising raising the osmotic pressure of the animal cell culture medium on a specific culture day during the cultivation of animal cells expressing the desired recombinant protein. .

In the present invention, the culture may be a fed-batch culture, but is not limited thereto.

In the present invention, the term "fed-batch culture" refers to a feeding medium continuously or discontinuously supplied to the culture at any time in the cell growth phase or the antibody production phase after starting culture with a basic medium or production medium. It means an ongoing cell culture.

In the present invention, "cell culture medium" or "culture medium" refers to a nutrient solution for maintaining, growing, proliferating or expanding cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture media can be optimized for specific cell culture, such as basic culture media formulated to support cell growth, or cell culture production media formulated to promote monoclonal antibody production, made by concentrating nutrients to high concentrations. There is a concentrated medium. The terms nutrient and medium components refer to components constituting the cell culture medium, and are used interchangeably in the present specification.

Specifically, the term "basic culture medium" or "basic medium" refers to a medium capable of supporting minimal cell growth. The basal medium supplies standard inorganic salts such as zinc, iron, magnesium, calcium and potassium, as well as trace elements, vitamins, energy sources, buffer systems and amino acids. Dulbecco's modified Eagle medium (DMEM), basic medium Eagle (BME), RPMI 1640, F-10, and F-12 medium are included in the basic medium, but are not limited thereto.

In addition, specifically, the term "cell culture production medium" or "production medium" refers to a medium used for the purpose of maximizing the expression of monoclonal antibodies in a bioreactor. The production medium can be the same as or different from the basic medium, and if different, it can be produced by concentrating the basic medium itself or adding specific ingredients to the basic medium.

The terms "feeding medium" and "additional culture medium" used in the present invention are a medium composed of a specific nutrient or a plurality of nutrients, and may all be concentrated products of the basic medium. Depending on the cells to be cultured, various components and concentrations of the feeding medium can be prepared.

In the culture process, the cell growth period refers to a period in which cell growth rapidly proceeds after inoculation. In the case of Chinese hamster ovary (CHO) cells, it is known that the number of cells increases most actively in the range of temperature range of 35 ~ 37℃ and pH range of 7.0 ~ 7.3. In the present invention, the cell growth phase culture parameter was a temperature of 36.5° C. and a pH of 7.1 or 7.0.

The term "inoculation" means injecting a medium to start cell culture and injecting cells into the injected medium. Here, the medium may be injected before the cells are injected or may be injected into the cell reactor at the same time as the cells. In large-scale animal cell culture, cells can be injected after the medium is usually injected in advance and the temperature and oxygen saturation are maintained within a predetermined range.

The term "antibody production phase" refers to the time when a change in the culture process is applied to maximize the production of monoclonal antibodies. Changes in the process to maximize production here include both lowering the temperature or dissolved oxygen, or changing the medium.

In the present invention, it was confirmed that the degree of change in galactoseization of the recombinant protein produced in animal cells expressing the recombinant protein may vary depending on the time point of increasing the osmotic pressure. That is, when the osmotic pressure is gradually increased during the incubation period, the degree of galactoseization does not differ significantly from the group that does not increase the osmotic pressure, whereas when the osmotic pressure is increased at a specific culture point during the incubation period, the G0F content increases significantly. By showing, it was confirmed that galactoseization can be controlled, specifically, galactoseization can be inhibited or reduced.

In addition, in the present invention, the step of raising the osmotic pressure is preferably performed during the main culture process of the animal cell expressing the desired recombinant protein, but is not limited thereto.

The term "main culture" as used herein refers to a culture step in which the actual production of monoclonal antibodies is performed. This is the last step of the culture process that started with one vial of cells, and when the main culture is finished, the process of purifying the produced monoclonal antibody continues. It can be distinguished from the term seed culture for the purpose of stepwise increasing the culture volume prior to the main culture.

The step of raising the osmotic pressure may be performed at a specific culture point during the main culture process of the animal cells, and specifically, performed on any one culture day from the 1st to the 10th day of the main culture based on the 0 day of the start of the main culture. May be, and more specifically, may be performed on any one of the 1st, 4th, 7th, and 10th day based on the 0 day of the start of the main culture, and more specifically, the 0 day of the start of the main culture. It may be performed on the 4th or 7th day, but is not limited thereto.

In addition, the step of increasing the osmotic pressure may be performed once during the culture process of the animal cell expressing the desired recombinant protein.

In addition, the step of raising the osmotic pressure is not particularly limited thereto, but may be performed so that the final osmotic pressure in the culture medium is 460 to 500 mOsm/kg, and more specifically, the increase in the osmotic pressure is the final osmotic pressure in the culture medium is 460 to It may be performed to be 480 mOsm/kg, but is not limited thereto. The final osmotic pressure in the culture medium refers to the osmotic pressure that appears at the end of the culture, but is not particularly limited thereto.

In addition, in the step of increasing the osmotic pressure, the target for increasing the osmotic pressure in the culture medium may be 400 to 440 mOsm/kg, more specifically 430 to 440 mOm/kg, but is not limited thereto. The target for increasing the osmotic pressure in the culture medium refers to the degree of the osmotic pressure to be increased at the time of performing the step of increasing the osmotic pressure, but is not particularly limited thereto.

In addition, the osmotic pressure increase may be performed by one or more methods selected from the group consisting of a method of supplementing sodium chloride or potassium chloride to the culture medium of the animal cells and a method of adding glucose to the culture medium, and more specifically, supplementing sodium chloride to the culture medium. It may be performed in a method, but is not limited thereto.

The method of supplementing the sodium chloride to the culture solution may be performed by increasing the osmotic pressure to a certain level by adding a 4M aqueous sodium chloride solution to the culture solution on a specific day during fed-batch culture. It is not limited thereto. When the osmotic pressure in the culture medium, and furthermore, in the cells is increased through the addition of an aqueous sodium chloride solution, the activity of beta-galactosidase increases, thereby inhibiting the galactosization of monoclonal antibodies.

In the present invention, the term "culture" or "culture medium" refers to a liquid containing cells contained in a shaker flask or a bioreactor in which culture is in progress. The culture and the culture medium can be used interchangeably. In addition, the culture medium and the culture medium can be classified according to the presence or absence of animal cells.

In the present invention, the recombinant protein may be an antibody, preferably a monoclonal antibody.

The term "monoclonal antibody" as used herein refers to an antibody that can be produced by a single antibody-forming cell, and recognizes one antigenic determinant.

The antibody of the present invention is not limited thereto, but preferably may include all therapeutic antibodies commonly used in the art, and specifically may be adalimumab.

In addition, the antibody is a concept that includes both full-length antibodies and antibody fragments, and the types of antibody fragments include all of Fv, Fab, Fab', F(ab') ₂ , Fd, and the like. The Fv includes both double disulfide Fv (dsFv) and short chain Fv (scFv) forms. Fd refers to the heavy chain portion contained in the Fab fragment.

Animal cells expressing the desired recombinant protein include, without limitation, natural or transformed cells expressing the recombinant protein. For the purposes of the present invention, it may be an animal cell capable of expressing a recombinant protein subject to regulation of galactose content, for example, a Chinese hamster ovary cell line (CHO) or a mouse myeloma cell line (NSO), but is limited thereto. no.

In addition, in order to control the galactoseization of the monoclonal antibody, optimization of culture parameters, concentration of a basic culture medium, and feeding of an optimized additional culture medium may be included.

In other words, after setting the range of galactosization of the desired monoclonal antibody, analyzing the galactosization of the monoclonal antibody expressed by the owned cell line, the method of controlling the galactoseization of the monoclonal antibody specified above according to the degree of demand for change of galactose is applied together. I can.

Specifically, examples of changes that can be implemented in the operation of the incubator for the production of monoclonal antibodies with controlled galactose are the optimization of culture parameters such as dissolved oxygen (DO), acidity (pH), culture temperature, and Agitation. . Specifically, in the cell growth period, the temperature was 36.5℃, dissolved oxygen was 30%, and the acidity was 7.0. In the antibody production period, the temperature was 30℃, dissolved oxygen was 30%, and the acidity was 7.0. In the method, it was confirmed that galactoseization of the monoclonal antibody can be controlled when incubated by changing the temperature in the antibody production phase to 28°C, dissolved oxygen to 20%, and acidification degree to 6.9.

In addition, in order to control galactose, the basic culture medium may be concentrated and used. Specifically, an animal-derived protein-free culture medium can be used, and a basic medium developed for fed-batch culture can be optimized to change the galactoseization of monoclonal antibodies. Specifically, when the basic culture medium was concentrated, specifically 0.8-fold or 1.4-fold concentrated medium was used in the main culture, it was possible to control the galactoseization of the monoclonal antibody.

In addition, it is possible to optimize the additional culture medium for the control of galactose. In the present invention, galactoseization was reduced by controlling the concentration of the additional culture medium and optimizing the concentration of metal components added to the additional culture medium.

Galactoseization of the desired recombinant protein prepared through the above method can be measured by N-glycan profile analysis using a Q-TOF or UPLC instrument. The information provided by the N-glycan profile analysis varies in G0F content, GI (Galactosylation Index), NGHC (non-glycosylated Heavy Chain), afucosylation percentage, and mannose content, but the present invention Mainly mentioned the change in the G0F content.

In addition, the method of the present invention may be for preparing a population of antibodies with reduced galactose. That is, after analyzing the galactoseization of the monoclonal antibody expressed by the owned cell line, it is a method that can be applied when an antibody population having a lower level of galactose than the analyzed value is to be prepared.

In the present invention, the term "antibody group" refers to a group of antibodies including antibodies with varying sugar chain contents, and for the purposes of the present invention, the antibody group includes galactosylated antibodies in a desired ratio, galactose It means a group of regulated antibodies.

Another aspect of the present invention is a method of regulating galactoseization of a desired recombinant protein, comprising raising the osmotic pressure of the animal cell culture medium during the culturing process of an animal cell expressing the desired recombinant protein.

The method and each term are as described above.

Specifically, the method may include raising the osmotic pressure of the animal cell culture medium on a specific culture day during the culture process of the animal cell expressing the desired recombinant protein, and specific details are as described above.

Another aspect of the present invention is a method for producing a recombinant protein of interest, in which galactose is regulated, comprising the step of supplementing asparagine to the animal cell culture medium during the cultivation of animal cells expressing the desired recombinant protein.

The terms described above are as described above.

In the present invention, it was confirmed that when asparagine was supplemented to the animal cell culture medium during the cultivation of animal cells expressing the desired recombinant protein, galactoseization of the desired recombinant protein could be regulated.

Specifically, the asparagine may be added in the form of an asparagine concentrate or a culture medium containing asparagine.

In addition, the supplementation of asparagine may be performed multiple times at a specific culture point during the process of culturing animal cells expressing the desired recombinant protein. Here, the supplementation of asparagine may be performed from 4 days to 10 days of culture based on the 0 day of the start of the main culture, and specifically, all of the 4 days, 7 days and 10 days of culture based on the 0 day of the start of the main culture. Thus, it is possible to gradually increase the concentration of asparagine in the culture medium.

When asparagine is supplemented in a single _{dose, the concentration of NH 4} ^{+ in the} culture medium is rapidly increased, which delays cell growth and eventually leads to a decrease in the final protein expression.However, according to the present invention, asparagine is added separately during feeding. In this case, there is an advantage of achieving a desired amount of protein production while controlling galactosization without such disadvantages.

The supplementation of asparagine may be performed so that the final concentration of asparagine in the culture medium of the animal cells is 27.6 to 33.6 mM.

Specifically, asparagine may be supplemented so that the final concentration of asparagine in the culture medium of animal cells is 33.6 mM, for example, asparagine may be supplemented so that the asparagine concentration in the culture medium of animal cells is additionally increased by 6 to 18 mM, As a more specific example, asparagine may be supplemented three times by sequentially adding the asparagine concentration in the culture medium of the animal cell by 6mM, 12mM, and 18mM, but is not limited thereto.

When this method is applied to animal cells, the _{ammonium ion concentration in the cell increases by inducing the production of ammonium ions (NH 4} ⁺ ), thereby increasing the pH of the trans Golgi body, thereby increasing the activity of beta-galactosyltransferase. Falling off may inhibit the galactoseization of monoclonal antibodies.

In addition, as described above, in order to control the galactoseization of the monoclonal antibody, optimization of culture parameters, concentration of a basic culture medium, and feeding of an optimized additional culture medium may be included.

In other words, after setting the range of galactosization of the desired monoclonal antibody, analyzing the galactosization of the monoclonal antibody expressed by the owned cell line, the method of controlling the galactoseization of the monoclonal antibody specified above according to the degree of demand for change of galactose is applied. I can.

Another aspect of the present invention is a method for regulating galactoseization of a desired recombinant protein, comprising the step of supplementing asparagine to the animal cell culture medium during the cultivation of an animal cell expressing the desired recombinant protein.

The method and each term are as described above.

Another aspect of the present invention is to increase the osmotic pressure of the animal cell culture medium during the cultivation of animal cells expressing the desired recombinant protein, and supplementing asparagine, wherein galactose is regulated, the desired recombinant protein. It is a manufacturing method of.

The terms described above are as described above.

In the present invention, when asparagine is added while increasing the osmotic pressure of the animal cell culture medium during the cultivation of animal cells expressing the desired recombinant protein monoclonal antibody, galactosization due to an increase in osmotic pressure is reduced, and at the same time, it decreases with an increase in osmotic pressure. It was confirmed that the content of the acidic isomeric antibody that can be increased by the addition of asparagine.

In the above method, the osmotic pressure increase and asparagine supplementation may be performed simultaneously or sequentially. For example, the osmotic pressure in the culture medium is increased by a method of supplementing sodium chloride, and asparagine is supplemented, or asparagine is supplemented, and then the osmotic pressure in the culture medium is increased by a method such as addition of sodium chloride, or asparagine supplementation and Increasing the osmotic pressure in the culture medium can be applied simultaneously.

Here, the osmotic pressure may be increased by applying the method described above.

The osmotic pressure increase may be performed so that the final osmotic pressure in the culture medium is 460 to 500 mOsm/kg, and the supplementation of asparagine may be supplemented with asparagine so that the final concentration of asparagine in the culture medium of the animal cell is 27.6 to 33.6 mM. have.

In addition, the recombinant protein may be an antibody, specifically a monoclonal antibody, and the content of the acidic isomer antibody of the antibody population prepared by the method at the same time as controlling the galactoseization of the desired recombinant antibody through the method of the present invention as described above. Can be adjusted. That is, the amount of acidic isomeric antibody that can decrease with increasing osmotic pressure and at the same time decreases galactose with increasing osmotic pressure can be increased by the addition of asparagine.

Accordingly, the use of the above method has the advantage of being able to control the degree of galactoseization of the target antibody population and at the same time control the content of the acidic isomer antibody of the target antibody population.

The acidic isomeric antibody is a kind of isomeric antibody. An isomeric antibody refers to an antibody in which some amino acids of the main active antibody have been modified by deamination or oxidation, and includes types of acidic isomeric antibodies and basic isomeric antibodies. For example, asparagine (asparagine) among amino acids is deaminated to become aspartate isomeric antibody, methionine (methionine) of amino acids is oxidized to methionine sulfate (methionine sulfate) isotropic antibody, and the like. In addition, when glutamate is present at the N-terminus of the heavy chain, the glutamate forms a pentagonal ring structure and includes an isomeric antibody modified with pyruglutamate.

Analysis of such an isomeric antibody can be performed using chromatography or the like, and in the present invention, it was performed by cation exchange resin chromatography analysis. Depending on the properties of monoclonal antibodies, the content of acidic, main, and basic isomers varies greatly. Depending on the culture conditions (culture parameters, production medium, etc.) of the cell line expressing the monoclonal antibody, the content of the charged isomer antibody of the monoclonal antibody may vary. Therefore, through the above method, it has the advantage of controlling the desired galactose content and simultaneously controlling the content of the acidic isomer antibody within a desired range.

The method of the present invention has the advantage of being able to effectively control the galactose content of the desired recombinant protein within a desired range. In addition, the method of the present invention using both the increase in osmotic pressure and the addition of asparagine has an effect of controlling not only galactose but also the content of acidic isomers, and thus can be effectively used in the preparation of a target antibody population.

Figure 1 shows the expression vector of adalimumab biosimilar.
Figure 2 shows the injection point of the sodium chloride aqueous solution during the fed-batch culture period.
3 shows the injection point of asparagine during the fed-batch culture period.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example 1: Control of galactoseization due to artificial increase in osmotic pressure

There are various methods of increasing the osmotic pressure of the culture medium during fed-batch culture. There is a method of adding an excess of glucose to the culture solution, a method of adding an aqueous potassium chloride or sodium chloride solution to the culture solution, and the like. In the present invention, as a representative method for increasing the osmotic pressure, a method of adding a 4M aqueous sodium chloride solution to the culture solution was applied.

Description of the manufacturing method and medium composition

In the present invention, the monoclonal antibody to which the osmotic pressure increase to the culture medium or the addition of asparagine is applied is an adalimumab biosimilar antibody, and adalimumab is a therapeutic agent for rheumatoid arthritis and Crohn's disease developed by abbott. After amplifying and generating adalimumab biosimilar DNA through polymerase chain reaction by referring to the amino acid sequences of the heavy and light chains of the adalimumab antibody of US 6,090,382, pCB using the promoter of pGL3 CUCBin, a vector developed by the company. -Am2.77_v5.4 (FIG. 1) was prepared, and it was transformed into a CHO-DXB11 cell line to produce an adalimumab biosimilar-expressing cell line. The company developed pCUCBin is one of the vectors mentioned in the Korean patent (a new fusion promoter and a recombinant vector containing the same, registration number 10-1038126).

The culture medium used in the present invention is three types of basic culture medium, production medium (main culture medium), and additional culture medium (feeding medium).

The basic culture medium is a medium used for the purpose of subculture. The production medium is a medium used for culture (main culture) performed for full-fledged antibody production after subculture, and can be prepared by enhancing specific components in the basic culture medium or adding new components. In the present invention, a 1.4-fold enhanced basic culture medium was used as the production medium. The additional culture medium is a culture medium added for the purpose of promoting cell growth and increasing the amount of antibody expression during the culture process. In the present invention, a 3.3-fold enhanced basic culture medium was used as an additional culture medium. The basic culture medium, production medium, and additional culture medium are all modified in IMDM (Iscove's Modified Dulbecco's Medium), and the components of the medium are shown in Table 1.

Basic culture medium components division ingredient IMDM modified medium Merck 100131 Buffer HEPES Buffer Sodium bicarbonate Carbon source Glucose Nitriogen source Glutamine Etc Sodium chloride Growth hormone Insulin Etc MTX Vitamin L-ascorbic acid Vitamin Biotin Vitamin Choline chloride Vitamin Folic acid Peptone Sheff CHO plus pf acf (Kerry) Metal Boric acid Metal Cobalt Chloride Hexahydrate Metal Copper Sulfate Pentahydrate Metal Ferric Citrate Metal Magnesium chloride anhydrous Metal Manganese sulfate monohydrate Metal Potassium nitrate Metal Sodium selenite pentahydrate Metal Zinc sulfate heptahydrate

The method for producing the antibody used in the present invention is an animal cell culture through a bioreactor, and the animal cell culture is a process in which animal cells are grown in a culture medium and the antibody is expressed through special measures (eg, lowering the temperature). The culture method is various such as batch culture, fed-batch culture, continuous culture, medium exchange culture, etc., but the culture method of the adalimumab biosimilar cell line used in the present invention is fed-batch culture. Fed-batch culture is a culture method in which culture is carried out with a production medium, and an additional culture medium is added once or twice or more on a specific day of culture.

The fed-batch culture method used in the Examples is to add an additional culture medium in an amount corresponding to 5% of the current culture medium volume of the production medium four times on days 1, 4, 7, and 10 of culture.

Example 1.1: Changes in galactoseization according to the time of osmotic pressure increase

A 250 mL shaker flask was used to actually conduct fed-batch culture at a scale of 30 mL. The feeding strategy of fed-batch culture and the time point of addition of aqueous sodium chloride solution were as shown in FIG. The culture started with an osmotic pressure of 320 mOsm/kg was fed with a 4M sodium chloride (NaCl) aqueous solution to artificially increase the osmotic pressure to 430 mOms/kg, resulting in a final osmotic pressure of 450 mOms/kg. The additional culture medium is first fed on the first day of cultivation, and after every 3 days, additional feeding is carried out for a total of 4 feedings, of which one or four times are divided (gradually increases the osmotic pressure during the culture period) 4 M sodium chloride (NaCl) aqueous solution was added to increase the osmotic pressure of the culture, followed by cultivation, and then the expression level and galactose of the monoclonal antibody were analyzed, and the results are shown in Table 2 below.

Changes in antibody quality according to the time of osmotic pressure increase Culture conditions Antibody expression level
(mg/L) G0F
(%)* Acidic
(%) Final osmotic pressure
(mOsm/kg) The osmotic pressure gradually increases during the incubation period. 1392.0 60.4 20.1 453.0 At the time of 1st feeding (1 day of culture)
Increase osmotic pressure 1215.5 66.1 17.6 445.0 2nd feeding (4 days of culture)
Increase osmotic pressure 1268.3 68.8 16.7 451.0 3rd feeding (7 days of culture)
Increase osmotic pressure 1438.7 67.1 16.4 451.0 4th feeding (10 days of culture)
Increase osmotic pressure 1580.1 66.2 17.4 454.0 Does not increase osmotic pressure 1771.8 63.5 18.5 364.0

*G0F(%) = G0F/(G0F+G1F+G2F)

As a result, as shown in Table 2, there was a difference in galactoseization of the monoclonal antibody according to the time point at which the 4M sodium chloride (NaCl) aqueous solution was fed. In the case of shaker flask culture, it is not easy to directly control dissolved oxygen (DO) and acidity (pH) during the culture process, so the difference in G0F between experimental conditions may not be large. However, in bioreactor culture, the dissolved oxygen and acidity can be directly controlled, so that the difference in G0F can be maximized.

Example 1.2: Monoclonal antibody galactose change according to the difference in osmotic pressure increase range

Example 1.1 showed that galactoseization of monoclonal antibodies may vary depending on the time point of increasing the osmotic pressure of the culture. When the aqueous sodium chloride solution was fed during the 2nd feeding (culture 4 days) and the 3rd feeding (culture 7 days) of the additional feeding medium, G0F increased to the maximum. Additionally, a second shaker flask fed-batch culture was performed to investigate the effect of the difference in the level of osmotic pressure increase in the culture on the galactoseization of the monoclonal antibody. The osmotic pressure at the beginning of the culture is 320 mOsm/kg, and according to the goal of increasing the osmotic pressure, a different amount of 4 M sodium chloride (NaCl) aqueous solution was added to the culture solution on the 4th or 7th day of culture, similar to the 1st shaker flask culture. Then, the expression level and galactoseization of the monoclonal antibody were analyzed.

Changes in antibody quality according to the difference in the range of osmotic pressure increase Culture conditions Osmotic pressure elevation target
(mOsm/kg) Antibody expression level
(mg/L) G0F
(%)* Acidic
(%) final
Osmotic pressure
(mOsm/kg) Osmotic pressure rise X X 1136.2 67.4 15.6 365.0 2nd feeding
(4 days of culture)
Increase osmotic pressure Up to 400 948.9 70.4 15.0 424.0 Up to 440 824.1 71.0 14.6 460.0 Up to 480 573.7 67.9 14.5 505.0 Up to 520 543.8 66.5 13.9 539.0 3rd feeding
(7 days of culture)
Increase osmotic pressure Up to 400 1053.4 70.5 14.9 416.0 Up to 440 927.4 69.5 14.5 454.0 Up to 480 851.1 68.5 14.3 497.0 Up to 520 775.1 66.9 14.2 533.0

*G0F(%) = G0F/(G0F+G1F+G2F)

The galactoseization of monoclonal antibodies varied according to the level of osmotic pressure increase in the culture medium. When the osmotic pressure of the culture was increased to 480 mOsm/kg or more during the cultivation process, the G0F content was rather decreased. In other words, there was an appropriate range of osmotic pressure to maximize the G0F content of monoclonal antibodies (up to 400 ~ 480 mOsm/kg during culture).

Example 1.3: Bioreactor (bioreactor) confirmation experiment

As a result of performing a confirmation experiment in a bioreactor based on the first and second shaker flask culture results, it was possible to reconfirm the change in galactoseization of the monoclonal antibody according to the conditions of increasing osmotic pressure.

At the actual 1 L scale in a 1.4 L bioreactor, at the time of secondary feeding (4 days of culture), each 4 M aqueous sodium chloride solution was added to the culture to artificially increase the osmotic pressure to 440, 500, 523 mOsm/kg, and then the culture was performed. Proceeded.

Changes in antibody quality according to the difference in the range of osmotic pressure increase in the bioreactor Conditions for increasing osmotic pressure (mOsm/kg) during secondary feeding Expression level
(mg/L) G0F
(%)* Acidic
(%) NH ₄ ⁺ concentration
(mM) Final osmotic pressure
(mOsm/kg) Does not rise 1980.6 63.7 26.5 3.6 321 Increase to 440 1740.2 71.8 18.4 7.7 479 Up to 500 1361.6 58.3 18.4 7.3 549 Rises to 523 1361.7 54.8 19.3 5.6 567

*G0F(%) = G0F/(G0F+G1F+G2F)

When the osmotic pressure of the culture medium was increased by 500 mOsm/kg or more during the secondary feeding (final osmotic pressure 549, 567 mOsm/kg), the G0F content decreased. In the case of a bioreactor experiment in which the osmotic pressure was raised to 440 mOsm/kg during the secondary feeding, the content of G0F was 71.8%.

Example 2: Production of excess ammonium in the culture medium through the addition of asparagine

According to the culture results of the present applicant (Table 4), when an excessive amount of asparagine was added to the culture solution, the concentration of ammonium ions (NH ₄ ⁺ ) in the culture solution increased dramatically. The galactosization of monoclonal antibodies can be controlled by _{indirectly increasing the concentration of NH 4} ⁺ in the culture medium through the addition of an excessive amount of asparagine.

Increase of ammonium with addition of asparagine to culture Culture conditions Added asparagine concentration
(mM) Final ammonium ion
(mM) No asparagine added. 0 3.4 Added asparagine 18.9 8.1

Example 2.1: Asparagine Concentrated solution added Federation culture

A 250 mL shaker flask was used to actually conduct fed-batch culture at a scale of 30 mL. The feeding strategy of fed-batch culture and the timing of injection of asparagine were as shown in FIG. 3. A concentrated solution of asparagine was prepared at 200 mM. The time of injecting the additional culture medium was culture 1, 4, 7, and 10 days, and the addition of the concentrated asparagine solution was performed every feeding (three times in total) from the second feeding time (4 days of culture). After incubation, the expression level of the monoclonal antibody and the galactoseization were analyzed.

Changes in Galactosization of Monoclonal Antibodies by Addition of Asparagine Culture conditions Antibody expression level
(mg/L) G0F
(%)* Acidic
(%) Final NH ₄ ⁺
(mM) Production medium Asparagine concentration added at one time feeding (mM) Added
Total asparagine concentration (mM) 1 X 0 0 1738.5 56.3 25.9 4.1 1 X One 3 1705.8 59.9 21.7 5.2 1 X 2 6 1535.2 61.4 19.2 6.7 1 X 4 12 1500.0 64.3 19.3 8.8 1.4 X 0 0 1729.9 60.2 21.1 5.6 1.4 X One 3 1721.0 61.0 21.3 7.4 1.4 X 2 6 1588.5 61.5 20.1 7.3 1.4 X 4 12 1563.7 61.9 19.3 9.0

*G0F(%) = G0F/(G0F+G1F+G2F)

The G0F content increased as asparagine was added to the culture medium by feeding. As the concentration of added asparagine increased, the final NH ₄ ⁺ concentration in the culture medium increased. Compared to the 1.4X production medium, the 1X production medium showed a greater increase in the G0F content for the input of asparagine. If asparagine was added at one time at the beginning of the culture, the NH ₄ ⁺ concentration increased from the beginning of the culture, delaying cell growth, and eventually lowering the final antibody expression level, so asparagine was divided and added during feeding.

Example 2.2. : Feeding of additional culture medium containing excess asparagine

Unlike the shaker flask fed-batch culture, in the bioreactor fed-batch culture, a concentrated asparagine solution was not separately prepared and included in an additional culture medium. An additional 6 mM of asparagine was added for each feeding, and an additional culture medium containing 120 mM asparagine was prepared, followed by feeding. After culturing at an actual 1 L scale in a 1.4 L bioreactor, the expression level of the monoclonal antibody and galactoseization were analyzed.

Bioreactor experiment using additional culture medium containing excess asparagine Culture conditions Antibody expression level
(mg/L) G0F
(%) Acidic
(%) NH ₄ ⁺
(mM) Final osmotic pressure
(mOsm/kg) No asparagine added 1980.6 63.7 26.5 3.6 321. From 2nd feeding to every feeding
6 mM asparagine added
(Total 18 mM added) 1281.8 73.9 20.6 13.5 379

The G0F content was increased by 10.2% in the batch using the additional culture medium to which an excess of asparagine was added. _{Considering that the NH 4} ⁺ concentration in the culture medium to which an excess of asparagine was added increases by 9.9 mM, it can be seen that the decrease in galactoseization of the monoclonal antibody is _{due to the change in the NH 4} ^{+ concentration in the culture medium.} The osmotic pressure was increased by 58 mOsm/kg in the culture medium to which asparagine was added in excess.

Example 3: Combination of artificially increasing osmotic pressure and addition of asparagine

During fed-batch culture, the G0F content of monoclonal antibodies could be increased by artificially increasing osmotic pressure or by adding asparagine. In order to investigate the effect of monoclonal antibodies on galactoseization and charge variants, a combination of artificial osmotic pressure increase and asparagine addition was applied.

Example 3.1: Bioreactor experiment in which osmotic pressure increase and asparagine addition were simultaneously applied

In a 1.4 L bioreactor, fed-batch culture was carried out by simultaneously applying an artificial increase in osmotic pressure or an increase in osmotic pressure and addition of asparagine at an actual 1 L scale. In the case of the bioincubator in which the osmotic pressure increase and the addition of asparagine were applied at the same time, the osmotic pressure of the culture was increased to 423 mOsm/kg in consideration of the effect of increasing the osmotic pressure by the addition of asparagine (120 mM asparagine was added to the additional culture medium). After incubation, the expression level of the monoclonal antibody and the galactoseization were analyzed.

Changes in galactoseization by simultaneous application of osmotic pressure increase and asparagine addition in bioreactor Culture conditions Antibody
Expression level
(mg/L) G0F
(%) Acidic (%) NH ₄ ⁺
(mM) Final osmotic pressure
(mOsm/kg) Increases osmotic pressure to 450 1511.6 73.6 20.6 8.80 502.0 Increases osmotic pressure to 423
+ Asparagine added 1548.1 75.4 23.1 10.5 490.0

Although the difference in G0F between the two conditions in which only the osmotic pressure was applied and the two conditions in which the osmotic pressure and asparagine were combined and applied to the process was not large, the G0F content was further increased. In the case of cultures to which asparagine was added, the specific gravity of acidic variant decreased compared to cultures without addition (Tables 6 and 7), but as a result of applying the combination of osmotic pressure and asparagine, acidic isomers were rather increased. I did.

In order to increase the G0F content, an artificial increase of the osmotic pressure, the addition of asparagine, the artificial increase of the osmotic pressure, and the addition of asparagine can be applied.In addition to controlling the G0F content, an acidic isomer antibody in the charge profile If you want to increase the proportion of the drug, a strategy of simultaneously increasing the osmotic pressure and adding asparagine will be useful.

From the above description, those skilled in the art to which the present invention pertains can understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed that all changes or modified forms derived from the meaning and scope of the claims to be described later, and equivalent concepts thereof, rather than the above detailed description, are included in the scope of the present invention.

Claims (27)

As a method for producing a recombinant antibody of interest, with reduced galactosylation, comprising the step of raising the osmotic pressure of the animal cell culture medium on a specific culture day during the cultivation of animal cells expressing the desired recombinant antibody,
The animal cell is a Chinese hamster ovary cell (CHO cell),
The step of raising the osmotic pressure is performed once on any one of the 1st, 4th, 7th and 10th day of culture based on the 0 day of the start of the main culture,
The increase of the osmotic pressure is performed so that the final osmotic pressure in the culture medium is 400 mOsm/kg to 500 mOsm/kg,
The reduction in galactosization is due to an increase in the G0F content, the method.
The method of claim 1, wherein the culture is a fed-batch culture.
delete delete The method of claim 1, wherein the step of raising the osmotic pressure is performed on the 4th or 7th day based on the 0 day of the start of the main culture.
The method of claim 1, wherein the osmotic pressure is increased so that the final osmotic pressure in the culture medium is 460 mOsm/kg to 500 mOsm/kg.
The method of claim 1, wherein the osmotic pressure increase is performed by at least one method selected from the group consisting of a method of supplementing sodium chloride or potassium chloride to the culture medium of the animal cells and a method of adding glucose to the culture medium.
The method of claim 1, wherein the antibody is adalimumab.
The method of claim 1, wherein the method is for preparing a population of antibodies with reduced galactoseization.
delete A method for reducing galactoseization of a recombinant antibody of interest, comprising raising the osmotic pressure of the animal cell culture medium on a specific culture day during the cultivation of animal cells expressing the desired recombinant antibody,
The animal cell is a Chinese hamster ovary cell (CHO cell),
The step of raising the osmotic pressure is performed on any one of the 1st, 4th, 7th and 10th days based on the 0 day of the start of the main culture
The increase of the osmotic pressure is performed so that the final osmotic pressure in the culture medium is 400 mOsm/kg to 500 mOsm/kg,
The reduction in galactosization is due to an increase in the G0F content, the method.
delete delete delete delete delete delete delete delete delete The method of claim 1, wherein the method further comprises the step of supplementing the animal cell culture medium with asparagine.
The method of claim 21, wherein the increase in osmotic pressure and supplementation of asparagine are performed simultaneously or sequentially.
The method of claim 21, wherein the increase in osmotic pressure is performed by at least one method selected from the group consisting of a method of supplementing sodium chloride or potassium chloride to the culture medium of the animal cells, and a method of adding glucose to the culture medium.
The method of claim 21, wherein the osmotic pressure is increased so that the final osmotic pressure in the culture medium is 460 mOsm/kg to 500 mOsm/kg.
The method of claim 21, wherein the supplementation of asparagine is supplemented with asparagine so that the final concentration of asparagine in the culture medium of the animal cell is 27.6 to 33.6 mM.
The method of claim 21, wherein the antibody is adalimumab.
The method of claim 21, wherein the method is for reducing galactoseization of the desired recombinant antibody and adjusting the content of the acidic isomer antibody in the antibody population prepared by the method.

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